This is an amended application for a competing renewal of a grant to study calcium homeostasis in neurons and glia. Calcium and sodium ions both play critical roles in the conduction and transmission of signals in the nervous system. In many types of cells, including neurons and glia, the concentrations of these two ions are linked by a transport system that exchanges sodium for calcium; calcium can be extruded or can enter the cells via this mechanism. The Na/Ca exchanger and the Na pump that sets up the Na electrochemical gradient to drive the exchanger are both found in great abundance in the nervous system. The investigator has focussed on the study of the Na/Ca exchanger in order to elucidate some of the critical factors that regulate intracellular Ca, both cytoplasmic Ca and particularly stored Ca. The PI's laboratory has shown that the Na/Ca exchanger has a significant influence on the amount of Ca stored in the endoplasmic reticulum (ER) and thus on the availability of intracellular Ca for cell signaling. Thus the principal investigator hypothesizes that the Na/Ca exchanger plays a critical role in the control of cell responsiveness in both neurons and glia. The goal of this project is to test this hypothesis and to elucidate some essential features of Ca homeostasis in astrocytes and neurons that relate to this central role of the Na/Ca exchanger. There are 4 specific aims. These include (1) To demonstrate the pivotal role of Na/Ca exchange in regulating intracellular Ca storage in cultured mouse astrocytes and neurons. (2) Determine the spatial distribution of the key Na and Ca transporters (Na/Ca exchange, Na and Ca pumps) and relate them to the sites of ER Ca storage. (3) Identify the Na/Ca exchange isoforms that are expressed in rat astrocytes and neurons and determine how the block of exchanger expression with antisense oligonucleotides affects Ca homeostasis, and (4) To complete the characterization of the kinetic properties of the Na/Ca exchanger in nerve terminals and determine the mechanisms involved in the modulation of this exchanger's kinetics. In summary, these studies will provide a detailed understanding of brain Na/Ca exchangers and their critical role in neuronal and glial Ca homeostasis. The results should also yield new insight into mechanisms that lead to Ca overload and neuronal and glial cell injury and death.